Methods of therapeutic monitoring of phenylacetic acid prodrugs

ABSTRACT

The present disclosure provides methods for adjusting the dosage of PAA prodrugs (e.g., HPN-100, PBA) based on measurement of PAA and PAGN in plasma and calculating the PAA:PAGN ratio so as to determine whether PAA to PAGN conversion is saturated.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/636,256, filed April 20, 2012, the disclosure of which isincorporated by reference herein in its entirety, including drawings.

BACKGROUND

Nitrogen retention disorders associated with elevated ammonia levelsinclude urea cycle disorders (UCDs), hepatic encephalopathy (HE), andadvanced kidney disease or kidney failure, often referred to asend-stage renal disease (ESRD).

UCDs include several inherited deficiencies of enzymes or transportersnecessary for the synthesis of urea from ammonia, including enzymesinvolved in the urea cycle. The urea cycle is depicted in FIG. 1, whichalso illustrates how certain ammonia-scavenging drugs act to assist inelimination of excessive ammonia. With reference to FIG. 1, N-acetylglutamine synthetase (NAGS)-derived N-acetylglutamate binds to carbamylphosphate synthetase (CPS), which activates CPS and results in theconversion of ammonia and bicarbonate to carbamyl phosphate. In turn,carbamyl phosphate reacts with ornithine to produce citrulline in areaction mediated by ornithine transcarbamylase (OTC). A second moleculeof waste nitrogen is incorporated into the urea cycle in the nextreaction, mediated by arginosuccinate synthetase (ASS), in whichcitrulline is condensed with aspartic acid to form argininosuccinicacid. Argininosuccinic acid is cleaved by argininosuccinic lyase (ASL)to produce arginine and fumarate. In the final reaction of the ureacycle, arginase (ARG) cleaves arginine to produce ornithine and urea. Ofthe two atoms of nitrogen incorporated into urea, one originates fromfree ammonia (NH₄ ⁺) and the other from aspartate. UCD individuals bornwith no meaningful residual urea synthetic capacity typically present inthe first few days of life (neonatal presentation). Individuals withresidual function typically present later in childhood or even inadulthood, and symptoms may be precipitated by increased dietary proteinor physiological stress (e.g., intercurrent illness). For UCD patients,lowering blood ammonia is the cornerstone of treatment.

HE refers to a spectrum of neurologic signs and symptoms believed toresult from hyperammonemia, which frequently occur in subjects withcirrhosis or certain other types of liver disease. HE is a commonmanifestation of clinically decompensated liver disease and mostcommonly results from liver cirrhosis with diverse etiologies thatinclude excessive alcohol use, hepatitis B or C virus infection,autoimmune liver disease, or chronic cholestatic disorders such asprimary biliary cirrhosis. Patients with HE typically show alteredmental status ranging from subtle changes to coma, features similar topatients with UCDs. It is believed that an increase in blood ammonia dueto dysfunctional liver in detoxifying dietary protein is the mainpathophysiology associated with HE (Ong 2003).

ESRD results from a variety of causes including diabetes, hypertension,and hereditary disorders. ESRD is manifested by accumulation in thebloodstream of substances normally excreted in the urine, including butnot limited to urea and creatinine. This accumulation in the bloodstreamof substances, including toxins, normally excreted in the urine isgenerally believed to result in the clinical manifestations of ESRD,sometimes referred to also as uremia or uremic syndrome. ESRD isordinarily treated by dialysis or kidney transplantation. To the extentthat urea, per se, contributes to these manifestations and thatadministration of a phenylacetic (PAA) prodrug may decrease synthesis ofurea (see, e.g., Brusilow 1993) and hence lower blood ureaconcentration, PAA prodrug administration may be beneficial for patientswith ESRD.

Subjects with nitrogen retention disorders whose ammonia levels and/orsymptoms are not adequately controlled by dietary restriction of proteinand/or dietary supplements are generally treated with nitrogenscavenging agents such as sodium phenylbutyrate (NaPBA, approved in theUnited States as BUPHENYL® and in Europe as AMMONAPS®), sodium benzoate,or a combination of sodium phenylacetate and sodium benzoate (AMMONUL®).These are often referred to as alternate pathway drugs because theyprovide the body with an alternate pathway to urea for excretion ofwaste nitrogen (Brusilow 1980; Brusilow 1991). NaPBA is a PAA prodrug.Another nitrogen scavenging drug currently in development for thetreatment of nitrogen retention disorders is glyceryltri-[4-phenylbutyrate] (HPN-100), which is described in U.S. Pat. No.5,968,979. HPN-100, which is commonly referred to as GT4P or glycerolPBA, is a prodrug of PBA and a pre-prodrug of PAA. The differencebetween HPN-100 and NaPBA with respect to metabolism is that HPN-100 isa triglyceride and requires digestion, presumably by pancreatic lipases,to release PBA (McGuire 2010), while NaPBA is a salt and is readilyhydrolyzed after absorption to release PBA.

HPN-100 and NaPBA share the same general mechanism of action: PBA isconverted to PAA via beta oxidation, and PAA is conjugated enzymaticallywith glutamine to form phenylacetylglutamine (PAGN), which is excretedin the urine. The structures of PBA, PAA, and PAGN are set forth below:

The clinical benefit of NaPBA and HPN-100 with regard to nitrogenretention disorders derives from the ability of PAGN to effectivelyreplace urea as a vehicle for waste nitrogen excretion and/or to reducethe need for urea synthesis (Brusilow 1991; Brusilow 1993). Because eachglutamine contains two molecules of nitrogen, the body rids itself oftwo waste nitrogen atoms for every molecule of PAGN excreted in theurine. Therefore, two equivalents of nitrogen are removed for each moleof PAA converted to PAGN. PAGN represents the predominant terminalmetabolite, and one that is stoichiometrically related to waste nitrogenremoval, a measure of efficacy in the case of nitrogen retention states.

In addition to nitrogen retention states, PAA prodrugs may be beneficialin a variety of other disorders for which PBA and/or PAA are believed tomodify gene expression and/or exert post-translational effects onprotein function. In the case of maple syrup urine disease (MSUD, alsoknown as branched-chain ketoaciduria), for example, the apparentlybeneficial effect of NaPBA in lowering plasma levels of branched chainamino acids is reported to be mediated by PBA-induced inhibition of thekinase that regulates activity of branched chain alpha-keto aciddehydrogenase complex or BCKDC. BCKDC is the enzyme that normally breaksdown branched-chain amino acids and is genetically defective in MSUDpatients (Bruneti-Pieri 2011). Similarly, the putative beneficialeffects of PAA prodrugs for the treatment of cancer (Chung 2000),neurodegenerative diseases (Ryu 2005), and sickle cell disease (Perrine2008) all involve alteration of gene expression and/orpost-translational effects on protein function via PBA and/or PAA.

Numerous publications reports adverse events following administration ofPBA and/or PAA (Mokhtarani 2012), and PAA is reported to causereversible toxicity when present in high levels in circulation. Whilemany of these publications have not recorded PAA blood levels and/ortemporally correlated adverse events with PAA levels, toxicities such asnausea, headache, emesis, fatigue, weakness, lethargy, somnolence,dizziness, slurred speech, memory loss, confusion, and disorientationhave been shown to be temporally associated with PAA levels ranging from499-285 μg/mL in cancer patients receiving PAA intravenously, and thesetoxicities have been shown to resolve with discontinuation of PAAadministration (Thiebault 1994; Thiebault 1995). Therefore, whenadministering PAA prodrugs for treatment of nitrogen retention disordersand other conditions, it is important to optimize dosing so as toachieve the desired therapeutic effect while minimizing the risk of PAAassociated toxicity.

SUMMARY

Provided herein is a clinically practical approach for utilizing andinterpreting blood levels of PAA and PAGN to adjust the dose of a PAAprodrug in order to minimize the risk of toxicities and maximize drugeffectiveness.

Provided herein in certain embodiments are methods of treating anitrogen retention disorder or a condition for which PAA prodrugadministration is expected to be beneficial in a subject comprising thesteps of administering a first dosage of a PAA prodrug, measuring plasmaPAA and PAGN levels, calculating a plasma PAA:PAGN ratio, anddetermining whether the PAA prodrug dosage needs to be adjusted based onwhether the PAA:PAGN ratio falls within a target range. In certainembodiments, the target range is 1 to 2.5, 1 to 2, 1 to 1.5, 1.5 to 2,or 1.5 to 2.5. In certain embodiments, a PAA:PAGN ratio above the targetrange indicates that the dosage of the PAA prodrug needs to bedecreased. In other embodiments, a PAA:PAGN ratio above the target rangeindicates that the dosage may need to be decreased, with the finaldetermination of whether to decrease the dosage taking into accountother characteristics of the subject such as biochemical profile orclinical characteristics such as target nitrogen excretion, actualnitrogen excretion, symptom severity, disorder duration, age, or overallhealth. In certain embodiments, a PAA:PAGN ratio below the target rangeindicates that the dosage of the PAA prodrug needs to be increased. Inother embodiments, a PAA:PAGN ratio below the target range indicatesthat the dosage may need to be increased, with the final determinationof whether to increase the dosage taking into account othercharacteristics of the subject such as biochemical profile or clinicalcharacteristics such as target nitrogen excretion, actual nitrogenexcretion, symptom severity, disorder duration, age, or overall health.In certain embodiments, a PAA:PAGN ratio that is within the target rangebut within a particular subrange (e.g., 1 to 1.5 or 2 to 2.5 where thetarget range is 1 to 2.5) indicates that the dosage of the PAA prodrugdoes not need to be adjusted, but that the subject needs to be subjectedto more frequent monitoring. In certain embodiments, the methods furthercomprise a step of administering an adjusted second dosage if such anadjustment is determined to be necessary based on the PAA:PAGN ratioand, optionally, other characteristics of the subject. In otherembodiments, the methods further comprise a step of administering asecond dosage that is the same as or nearly the same as the first dosageif no adjustment in dosage is deemed to be necessary. In certainembodiments, the nitrogen retention disorder is UCD, HE, or ESRD. Incertain embodiments, the condition for which PAA prodrug administrationis expected to be beneficial is cancer, a neurodegenerative diseases, ametabolic disorder, or sickle cell disease. In certain embodiments, thePAA prodrug is HPN-100 or NaPBA. In certain embodiments, measurement ofplasma PAA and PAGN levels takes place after the first dosage of the PAAprodrug has had sufficient time to reach steady state, such as at 48hours to 1 week after administration.

Provided herein in certain embodiments are methods of treating anitrogen retention disorder or a condition for which PAA prodrugadministration is expected to be beneficial in a subject who haspreviously received a first dosage of PAA prodrug comprising the stepsof measuring plasma PAA and PAGN levels, calculating a plasma PAA:PAGNratio, and determining whether the PAA prodrug dosage needs to beadjusted based on whether the PAA:PAGN ratio falls within a targetrange. In certain embodiments, the target range is 1 to 2.5, 1 to 2, 1to 1.5, 1.5 to 2, or 1.5 to 2.5. In certain embodiments, a PAA:PAGNratio above the target range indicates that the dosage of the PAAprodrug needs to be decreased. In other embodiments, a PAA:PAGN ratioabove the target range indicates that the dosage may need to bedecreased, with the final determination of whether to decrease thedosage taking into account other characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health. In certain embodiments, a PAA:PAGNratio below the target range indicates that the dosage of the PAAprodrug needs to be increased. In other embodiments, a PAA:PAGN ratiobelow the target range indicates that the dosage may need to beincreased, with the final determination of whether to increase thedosage taking into account other characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health. In certain embodiments, a PAA:PAGNratio that is within the target range but within a particular subrange(e.g., 1 to 1.5 or 2 to 2.5 where the target range is 1 to 2.5)indicates that the dosage of the PAA prodrug does not need to beadjusted, but that the subject needs to be subjected to more frequentmonitoring. In certain embodiments, the methods further comprise a stepof administering an adjusted second dosage if such an adjustment isdetermined to be necessary based on the PAA:PAGN ratio and, optionally,other characteristics of the subject. In other embodiments, the methodsfurther comprise a step of administering a second dosage that is thesame as or nearly the same as the first dosage if no adjustment indosage is deemed to be necessary. In certain embodiments, the nitrogenretention disorder is UCD, HE, or ESRD. In certain embodiments, thecondition for which PAA prodrug administration is expected to bebeneficial is cancer, a neurodegenerative diseases, a metabolicdisorder, or sickle cell disease. In certain embodiments, measurement ofplasma PAA and PAGN levels takes place after the first dosage of the PAAprodrug has had sufficient time to reach steady state, such as at 48hours to 1 week after administration.

Provided herein in certain embodiments are methods of adjusting thedosage of a PAA prodrug to be administered to a subject comprising thesteps of administering a first dosage of a PAA prodrug, measuring plasmaPAA and PAGN levels, calculating a plasma PAA:PAGN ratio, anddetermining whether the PAA prodrug dosage needs to be adjusted based onwhether the PAA:PAGN ratio falls within a target range. In certainembodiments, the target range is 1 to 2.5, 1 to 2, 1 to 1.5, 1.5 to 2,or 1.5 to 2.5. In certain embodiments, a PAA:PAGN ratio above the targetrange indicates that the dosage of the PAA prodrug needs to bedecreased. In other embodiments, a PAA:PAGN ratio above the target rangeindicates that the dosage may need to be decreased, with the finaldetermination of whether to decrease the dosage taking into accountother characteristics of the subject such as biochemical profile orclinical characteristics such as target nitrogen excretion, actualnitrogen excretion, symptom severity, disorder duration, age, or overallhealth. In certain embodiments, a PAA:PAGN ratio below the target rangeindicates that the dosage of the PAA prodrug needs to be increased. Inother embodiments, a PAA:PAGN ratio below the target range indicatesthat the dosage may need to be increased, with the final determinationof whether to increase the dosage taking into account othercharacteristics of the subject such as biochemical profile or clinicalcharacteristics such as target nitrogen excretion, actual nitrogenexcretion, symptom severity, disorder duration, age, or overall health.In certain embodiments, a PAA:PAGN ratio that is within the target rangebut within a particular subrange (e.g., 1 to 1.5 or 2 to 2.5 where thetarget range is 1 to 2.5) indicates that the dosage of the PAA prodrugdoes not need to be adjusted, but that the subject needs to be subjectedto more frequent monitoring. In certain embodiments, the methods furthercomprise a step of administering an adjusted second dosage if such anadjustment is determined to be necessary based on the PAA:PAGN ratioand, optionally, other characteristics of the subject. In otherembodiments, the methods further comprise a step of administering asecond dosage that is the same as or nearly the same as the first dosageif no adjustment in dosage is deemed to be necessary. In certainembodiments, measurement of plasma PAA and PAGN levels takes place afterthe first dosage of the PAA prodrug has had sufficient time to reachsteady state, such as at 48 hours to 1 week after administration.

Provided herein in certain embodiments are methods of determiningwhether a first dosage of a PAA prodrug can be safely administered to asubject comprising the steps of administering the first dosage of a PAAprodrug, measuring plasma PAA and PAGN levels, calculating a plasmaPAA:PAGN ratio, and determining whether the first dosage can be safelyadministered based on whether the PAA:PAGN ratio falls above a targetrange. In certain embodiments, the target range is 1 to 2.5, 1 to 2, 1to 1.5, 1.5 to 2, or 1.5 to 2.5. In certain embodiments, a PAA:PAGNratio above the target range indicates that the first dosage is unsafeand needs to be decreased. In other embodiments, a PAA:PAGN ratio abovethe target range indicates that the first dosage is potentially unsafeand may need to be decreased, with the final determination of whether todecrease the dosage taking into account other characteristics of thesubject such as biochemical profile or clinical characteristics such astarget nitrogen excretion, actual nitrogen excretion, symptom severity,disorder duration, age, or overall health. In certain embodiments, aPAA:PAGN ratio that is within the target range but within a particularsubrange (e.g., 2 to 2.5 where the target range is 1 to 2.5) indicatesthat the first dosage is likely safe, but that the subject needs to besubjected to more frequent monitoring. In certain embodiments, themethods further comprise a step of administering an adjusted seconddosage if such an adjustment is determined to be necessary based on thePAA:PAGN ratio and, optionally, other characteristics of the subject. Incertain embodiments, measurement of plasma PAA and PAGN levels takesplace after the first dosage of the PAA prodrug has had sufficient timeto reach steady state, such as at 48 hours to 1 week afteradministration.

Provided herein in certain embodiments are methods of determiningwhether a first dosage of a PAA prodrug is likely to be effective fortreating a nitrogen retention disorder or another disorder for which PAAprodrug administration is expected to be beneficial comprising the stepsof administering the first dosage of a PAA prodrug, measuring plasma PAAand PAGN levels, calculating a plasma PAA:PAGN ratio, and determiningwhether the first dosage is likely to be effective based on whether thePAA:PAGN ratio falls below a target range. In certain embodiments, thetarget range is 1 to 2.5, 1 to 2, 1 to 1.5, 1.5 to 2, or 1.5 to 2.5. Incertain embodiments, a PAA:PAGN ratio below the target range indicatesthat the first dosage is unlikely to be effective needs to be increased.In other embodiments, a PAA:PAGN ratio below the target range indicatesthat the first dosage is potentially ineffective and may need to beincreased, with the final determination of whether to increase thedosage taking into account other characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health. In certain embodiments, a PAA:PAGNratio that is within the target range but within a particular subrange(e.g., 1 to 1.5 where the target range is 1 to 2.5) indicates that thefirst dosage is likely effective, but that the subject needs to besubjected to more frequent monitoring. In certain embodiments, themethods further comprise a step of administering an adjusted seconddosage if such an adjustment is determined to be necessary based on thePAA:PAGN ratio and, optionally, other characteristics of the subject. Incertain embodiments, measurement of plasma PAA and PAGN levels takesplace after the first dosage of the PAA prodrug has had sufficient timeto reach steady state, such as at 48 hours to 1 week afteradministration.

In certain embodiments, methods are provided for optimizing thetherapeutic efficacy of a PAA prodrug in a subject who has previouslybeen adminsitered a first dosage of PAA prodrug comprising the steps ofmeasuring plasma PAA and PAGN levels, calculating a plasma PAA:PAGNratio, and determining whether the PAA prodrug dosage needs to beadjusted based on whether the PAA:PAGN ratio falls within a targetrange. In certain embodiments, the target range is 1 to 2.5, 1 to 2, 1to 1.5, 1.5 to 2, or 1.5 to 2.5. In certain embodiments, a PAA:PAGNratio above the target range indicates that the dosage of the PAAprodrug needs to be decreased. In other embodiments, a PAA:PAGN ratioabove the target range indicates that the dosage may need to bedecreased, with the final determination of whether to decrease thedosage taking into account other characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health. In certain embodiments, a PAA:PAGNratio below the target range indicates that the dosage of the PAAprodrug needs to be increased. In other embodiments, a PAA:PAGN ratiobelow the target range indicates that the dosage may need to beincreased, with the final determination of whether to increase thedosage taking into account other characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health. In certain embodiments, a PAA:PAGNratio that is within the target range but within a particular subrange(e.g., 1 to 1.5 or 2 to 2.5 where the target range is 1 to 2.5)indicates that the dosage of the PAA prodrug does not need to beadjusted, but that the subject needs to be subjected to more frequentmonitoring. In certain embodiments, the methods further comprise a stepof administering an adjusted second dosage if such an adjustment isdetermined to be necessary based on the PAA:PAGN ratio and, optionally,other characteristics of the subject. In other embodiments, the methodsfurther comprise a step of administering a second dosage that is thesame as or nearly the same as the first dosage if no adjustment indosage is deemed to be necessary. In certain embodiments, measurement ofplasma PAA and PAGN levels takes place after the first dosage of the PAAprodrug has had sufficient time to reach steady state, such as at 48hours to 1 week after administration.

In certain embodiments, methods are provided for obtaining a plasmaPAA:PAGN ratio within a target range in a subject comprising the stepsof administering a first dosage of a PAA prodrug, measuring plasma PAAand PAGN levels, calculating a plasma PAA:PAGN ratio, and determiningwhether the PAA:PAGN ratio falls within the target range. If thePAA:PAGN ratio does not fall within the target range, an adjusted seconddosage is administered, and these steps are repeated until a plasmaPAA:PAGN ratio falling within the target range is achieved. In certainembodiments, the target range is 1 to 2.5, 1 to 2, 1 to 1.5, 1.5 to 2,or 1.5 to 2.5. In certain embodiments, a PAA:PAGN ratio above the targetrange indicates that the dosage of the PAA prodrug needs to be decreasedand a PAA:PAGN ratio below the target range indicates that the dosage ofthe PAA prodrug needs to be increased. In certain embodiments,measurement of plasma PAA and PAGN levels takes place after the firstdosage of the PAA prodrug has had sufficient time to reach steady state,such as at 48 hours to 1 week after administration.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Urea cycle.

FIG. 2: Plasma PAA levels versus plasma PAA:PAGN ratio in (A) allsubjects combined (healthy adults, patients age 2 months and above withUCDs, and patients with cirrhosis), (B) patients age 2 months and abovewith UCDs, and (C) patients with cirrhosis.

FIG. 3: Estimated probability (95% confidence interval (c.i.)) ofcorrectly detecting elevated plasma PAA:PAGN ratio (≧2.0) with a singleblood sample at a designated time.

FIG. 4: Distribution of plasma PAA:PAGN ratio (log scale) by time sincedosing (hours) and category of maximum PAA:PAGN ratio in all subjectscombined.

FIG. 5: Distribution of plasma PAA concentrations (μg/mL) by PAA:PAGNratio for (A) all subjects and (B) UCD and HE subjects.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

The enzymes responsible for beta oxidation of PBA to PAA are present inmost cell types capable of utilizing fatty acids as energy substrates,and the widespread distribution of these enzymes presumably accounts forthe rapid and essentially complete conversion of PBA to PAA. However,the enzymes that conjugate PAA with glutamine to form PAGN are foundprimarily in the liver and to a lesser extend in kidneys (Moldave 1957).Therefore, the conversion of PAA to PAGN may be affected under severalcircumstances, including the following: a) if conjugation capacity issaturated (e.g., by high doses of PAA prodrug); b) if conjugationcapacity is compromised (e.g., by severe hepatic and/or renaldysfunction); c) if the substrate (glutamine) for PAA to PAGNconjugation is rate limiting; d) genetically determined variability(i.e., polymorphisms) in the enzymes responsible for PAA to PAGNconversion, or e) in young children, since the capacity to convert PAAto PAGN varies with body size measured as body surface area (Monteleone2012). The presence of any one of these conditions may lead toaccumulation of PAA in the body, which causes reversible toxicity.

The goal of PAA prodrug administration in subjects with nitrogenretention disorders is to provide a sufficient dosage to obtain adesired level of nitrogen removal while avoiding excess build-up of PAA.The goal of PAA prodrug administration in patients without a nitrogenretention disorder (e.g., a neurodegenerative disease) is to achievecirculating metabolite levels necessary to produce a clinical benefit byalteration of gene expression and/or protein folding or function.However, there are several difficulties associated with determining theproper dosage in patients with nitrogen retention disorders.

Plasma PAA and PAGN levels are affected by various factors, includingtiming of the blood draw in relation to drug administration, hepaticfunction, availability of metabolizing enzymes, and availability ofsubstrates required for metabolism. A random PAA level drawn during anoutpatient visit to determine if levels are in the toxicity rangewithout considering concomitant PAGN level is insufficient to informdosing. First, PAA levels vary many-fold over the course of the day,fluctuating a great deal between peak and trough levels. For example, inthe Hyperion pivotal study evaluating HPN-100 for use in treating adultUCD (Study ID HPN-100-006, Clinical Trials ID NCT00992459), serial bloodsamples were obtained for PK studies over a 24 hour period during whichsubjects were receiving HPN-100 or NaPBA. The fluctuation index for PAAover a 24 hour period, which represents the fluctuation between maximumconcentration (typically observed after the last daily dose or atapproximately 12 hours) and minimum concentration (typically observed inthe morning after overnight fasting or at 0 hours), indicated a veryhigh degree of variability (2150% for NaPBA and 1368% for HPN-100).Therefore, a single plasma PAA level may not be representative of thehighest PAA level a patient may experience during the day. Second, ahigh plasma PAA level may only be indicative of the high doses a subjectis receiving rather than a point of concern if the subject iseffectively conjugating PAA with glutamine to form PAGN. Therefore,basing dose adjustment on only on a high PAA level without consideringconcomitant plasma PAGN level may result in unnecessary dose reductionand under-treatment of the patient. Conversely, a PAA level seeminglybelow the levels associated with toxicity might be taken as anindication of satisfactory dosing without appreciating the fact that theconcomitant PAGN level may not be proportional to PAA, indicating thatPAA is not being efficiently utilized and may be accumulating.

Previous studies have shown that conversion of PAA to PAGN is asaturable process that varies considerably among individuals (see, e.g.,Monteleone 2012), and that patients with hepatic impairment have higherPAA levels than patients without hepatic impairment (Ghabril et al.,“Glycerol phenylbutyrate (GPD) administration in patients with cirrhosisand episodic hepatic encephalopathy (HE),” submitted to DigestiveDisease Week, 2012). If PAGN formation is affected by any of the abovefactors, PAA will be accumulated and waste nitrogen may not be removedfrom the body. Previous studies have also shown that a small proportionof individuals, including both healthy adults ad patients with UCDs orHE, have higher PAA levels than the remainder of the population,presumably due to individual differences in conjugating PAA to PAGN, andthat PAA levels fluctuate many-fold during the day depending on the doseand the timing of blood sample relative to the last dose so that asingle plasma level may not be informative (Lee 2010; Lichter 2011).

Although the goal of PAA prodrug therapy for nitrogen retentiondisorders is to achieve ammonia levels within a normal limit, there isno correlation between plasma PAA levels and blood ammonia. Nitrogenretention disorder subjects are normally “dosed to effect,” meaning thatsubjects with absent or severely deficient urea synthetic capacityrequire higher doses of PAA prodrugs than do mildly deficient UCDpatients. These higher dosages are generally associated with higher PAAlevels, such that the conventional PK/PD response (higher active moiety,i.e., PAA, correlates with lower harmful substance, i.e., ammonia) doesnot apply. Therefore, there is no single target plasma PAA level thatcan be applied to patients with UCDs or other nitrogen retentiondisorders based on their blood ammonia.

Patients with severe hepatic impairment are at increased risk of PAAaccumulation due to inadequate levels of PAA conjugating enzymes iftreated with PAA-prodrugs. UCD patients without hepatic impairment whosePAA conjugating enzymes are readily saturated are also at increased riskof PAA accumulation if treated with PAA-producing compounds. Otherpatients without nitrogen retention are at increased risk of PAAaccumulation due to limited availability of glutamine as the substrateto form PAGN if treated with PAA-producing compounds, which accumulatesin patients with nitrogen retention states.

WO09/134460 and WO10/025303 disclose methods for determining aneffective dosage of a PAA prodrug based on urinary PAGN levels, whichwas found to be a more reliable indictor of effective dosage than plasmalevels of PAA or other metabolites. Although such measurements arehighly useful for evaluating waste nitrogen removal, they do not providecomplete information regarding a subject's ability to utilize theprodrug.

Since PAA, PAGN, and ammonia levels do not provide the informationnecessary to determine whether a subject is effectively converting PBAto PAGN (i.e., effectively utilizing the PAA prodrug), there is a needfor improved methods of adjusting PAA prodrug dosage and incorporatingsuch adjustments into methods of treating nitrogen retention disorders.

As disclosed herein, plasma PAA:PAGN ratio has been found to provide anunexpectedly accurate measure of PAA prodrug metabolism in subjects withnitrogen retention disorders and/or hepatic impairment. It was foundthat subjects who can readily convert PAA to PAGN and have not reachedthe saturation point with respect to PAA to PAGN conversion will have aplasma PAA:PAGN ratio of 2.5 or below (when both are measured in μg/mL),and that subjects with PAA:PAGN ratios above 2.5 have a significantlyhigher chance of experience a PAA level above 400 μg/mL or 500 μg/mLover a 24 hour period. A PAA/PAGN ratio of less than 2.5 was associatedprimarily with healthy adult or adolescent subjects and normal liverfunction, with subjects having a ratio below 2.5 exhibiting a 1%probability of experiencing a PAA level greater than 400 μg/mL andalmost no chance of exhibiting a PAA level greater than 500 μg/mL at anypoint during a 24 hour period. A ratio greater than 2.5, on the otherhand, was generally seen in subjects with moderate hepatic impairment, asubset of healthy subjects or UCD patients with relatively lowersaturation point and difficulty conjugating PAA to form PAGN, andpatients with a low body surface area. Subjects with a ratio greaterthan 2.5, on the other hand, exhibited a 20-36% likelihood ofexperiencing a PAA level greater than 400 μg/mL during the day, and anapproximately 10% likelihood of experiencing a PAA level of 500 μg/L orgreater. In subjects with a ratio greater than 3, the likelihood ofexperiencing a PAA level higher than 500 μg/mL increased to as high as25%. These results show that a plasma PAA:PAGN ratio exceeding 2.5 in apatient with unexplained neurological adverse events and normal ammoniaindicates that dosage adjustment should be considered. Thus, plasmaPAA:PAGN ratio provides a clinically useful surrogate for evaluating theefficiency of PAA to PAGN conversion.

Plasma PAA:PAGN ratio indicates whether a PAA prodrug is beingeffectively utilized and scavenging nitrogen, and therefore provides anindirect and simple measure of saturation of conjugating enzymes,availability of substrate, and possible effect of hepatic or renalimpairment on this process. Calculating this ratio will allow effectivetreatment and dose adjustment in subjects with known hepatic impairment,subjects presenting with signs and symptoms overlapping betweenhyperammonemia and PAA toxicities, and subjects who are not clinicallycontrolled despite increasing the dosage of drugs.

One of ordinary skill in the art would generally not consider the ratioof an active metabolite such as PAA to a terminal metabolite such asPAGN when making therapeutic decisions because they would expect thathigher levels of the active metabolite would result in a proportionatelyhigher response (as measured by PAGN production) and increased efficacy(i.e., waste nitrogen removal). However, the results provided hereinshow that the use of plasma PAA:PAGN ratios to evaluate and adjust PAAprodrug dosage is unexpectedly superior to the use of PAA or PAGN levelsalone. Once a subject exceeds a specific PAA:PAGN ratio, there is a highlikelihood that they are not effectively utilizing the active moiety andthat further increasing PAA prodrug dosage may not increase efficacy andmay actually result in PAA accumulation and toxicity.

Based on these findings, methods are provided herein for treatingnitrogen retention disorders and evaluating and adjusting the dosage ofa PAA prodrug based on plasma PAA:PAGN ratio. Generally, these methodscomprise steps of measuring plasma PAA and PAGN levels, calculating thePAA:PAGN ratio, and determining whether the ratio falls within a targetrange, with this determination being used at least in part to decidewhether to adjust PAA prodrug dosage. In these methods, PAA:PAGN ratiocan be used to ensure that urinary PAGN output, plasma ammoniaconcentration, and/or PAA levels fall within a predefined target range.Such methods represent an improvement over previously developed methodsfor evaluating PAA prodrug dosage and efficacy in that they allow formore accurate dosing, greater efficacy, and decreased risk of toxicityassociated with PAA accumulation.

Disclosed herein are target ranges for the ratio of plasma PAA to PAGNin subjects who are receiving PAA prodrug therapy. In certainembodiments, a subject exhibiting a PAA:PAGN ratio falling within atarget range is classified as properly dosed, meaning that they do notrequire a PAA prodrug dosage adjustment, while a subject exhibiting aPAA:PAGN ratio falling outside the target range is classified asimproperly dosed, meaning that they require an adjustment in PAA prodrugdosage. In certain of these embodiments, a subject exhibiting a plasmaPAA:PAGN ratio falling above a target range is classified as requiring adecreased dosage of PAA prodrug, while a subject exhibiting a plasmaPAA:PAGN ratio falling below a target range is classified as requiringan increased dosage of PAA prodrug. In other embodiments, a subjectexhibiting a plasma PAA:PAGN ratio falling above a target range isclassified as requiring a decreased dosage of PAA prodrug, while asubject exhibiting a plasma PAA:PAGN ratio falling below a target rangeis classified as potentially requiring an increase in PAA prodrugdosage. In still other embodiments, a subject exhibiting a plasmaPAA:PAGN ratio falling above a target range is classified as potentiallyrequiring a decreased dosage of PAA prodrug, while a subject exhibitinga plasma PAA:PAGN ratio falling below a target range is classified aspotentially requiring an increase in PAA prodrug dosage. In thoseembodiments where a subject is classified as potentially requiring anincrease or decrease in PAA prodrug dosage based on their PAA:PAGNratio, a decision as to whether to increase or decrease dosage may bebased on one or more additional characteristics of the subject such asbiochemical profile or clinical characteristics such as target nitrogenexcretion, actual nitrogen excretion, symptom severity, disorderduration, age, or overall health.

In certain embodiments, the target range for plasma PAA:PAGN ratio is 1to 2.5, meaning that a subject exhibiting a PAA:PAGN falling within thisrange is classified as properly dosed. In other embodiments, the targetrange for plasma PAA:PAGN ratio is 1 to 2, 1 to 1.5, 1.5 to 2, or 1.5 to2.5. In certain of those embodiments where the target range is 1 to 2.5,a subject with a PAA:PAGN ratio above 2.5 is classified as requiring adecrease in PAA prodrug dosage, while a subject with a PAA:PAGN ratiofalling below 1 is classified as potentially requiring an increase inPAA prodrug dosage. In certain of these embodiments, a subject isnecessarily classified as requiring an increase in PAA prodrug dosage iftheir ratio is below 1. In other embodiments, a subject with a PAA:PAGNratio of less than 1 is only classified as requiring an increase in PAAprodrug dosage if one or more additional clinical or biochemicalcharacteristics are satisfied (e.g., the subject is exhibiting severesymptoms of a nitrogen retention disorder).

In certain embodiments, the target range for plasma PAA:PAGN ratio maycomprise one or more subranges, with subjects falling within differentsubranges being treated differently despite falling within the targetrange. For example, where a target range is 1 to 2.5, a subjectexhibiting a PAA:PAGN ratio below 1 or above 2.5 may be classified asrequiring an adjustment in PAA prodrug dosage. Within the target range,subjects with a PAA:PAGN ratio falling within a particular subrange maybe treated as properly dosed, improperly dosed (i.e., requiring a dosageadjustment), or properly dosed but requiring more frequent monitoring.For example, subjects having a PAA:PAGN ratio greater than 2 but notgreater than 2.5 may be classified as properly dosed but requiring morefrequent monitoring.

In certain embodiments, subrange boundaries or the treatment of subjectsfalling within a particular subrange will depend in part on a subject'sspecific characteristics, including for example biochemical profile orclinical characteristics such as target nitrogen excretion, actualnitrogen excretion, symptom severity, disorder duration, age, or overallhealth. For example, in certain embodiments a first subject with aPAA:PAGN ratio falling within the subrange of 2 to 2.5 may be classifiedas properly dosed but requiring frequent monitoring, while a secondsubject falling within the same subrange may be classified as requiringa decreased dosage of PAA prodrug. Similarly, a first subject with aPAA:PAGN ratio falling within the subrange of 1 to 1.5 may be classifiedas properly dosed but requiring frequent monitoring, while a secondsubject falling within the same subrange may be classified as requiringan increased dosage of PAA prodrug. For example, a subject who hasrecently exhibited particularly acute symptoms associated with aparticular disorder may be classified as requiring an increased dosageof PAA prodrug when exhibiting a PAA:PAGN ratio of 1 to 1.5, while asubject who is clinically controlled may be classified as properly doseddespite a ratio falling within the same subrange.

In certain embodiments, methods are provided herein for treating anitrogen retention disorder or a condition for which PAA prodrugadministration is expected to be beneficial in a subject that haspreviously received a first dosage of a PAA prodrug. These methodscomprise measuring plasma PAA and PAGN levels, calculating the plasmaPAA:PAGN ratio, determining whether the PAA prodrug dosage needs to beadjusted based on whether the PAA:PAGN ratio falls within a targetrange, and administering a second dosage of the PAA prodrug. In certainembodiments, the target range for PAA:PAGN ratio is 1 to 2.5 or 1 to 2.In certain of these embodiments, the second dosage is greater than thefirst dosage if the PAA:PAGN ratio is less than 1 (i.e., the dosage isincreased) and less than the first dosage if the PAA:PAGN ratio isgreater than 2.5 (i.e., the dosage is decreased). In other embodiments,the second dosage may or may not be greater than the first dosage if thePAA:PAGN ratio is less than 1, depending on one or more othercharacteristics of the subject. In certain embodiments, the seconddosage is equal to the first dosage when the PAA:PAGN ratio is 1 to 2.5,i.e., falling within the target range. In certain embodiments, thetarget range is divided into one or more subranges. In certain of theseembodiments, the second dosage may be equal to the first dosage if thePAA:PAGN ratio is 1 to 1.5 or 2 to 2.5, but the subject may be subjectedto more frequent monitoring. In certain other embodiments, the seconddosage may be greater than the first dosage if the PAA:PAGN ratio is 1to 1.5 or 1 to 2 and the subject has recently exhibited particularlyacute symptoms of a nitrogen retention disorder or another condition forwhich PAA prodrug administration is expected to be beneficial.Similarly, the second dosage may be less than the first dosage if thePAA:PAGN ratio is greater than 1.5 or 2 but not greater than 2.5,depending on the subject's specific characteristics. In certainembodiments, the increase or decrease in the second dosage versus thefirst dosage depends on the precise plasma PAA:PAGN ratio. For example,where the plasma PAA:PAGN ratio is only slightly less than 1, the dosagemay be increased only slightly, but where the PAA:PAGN ratio issignificantly less than 1, the dosage may be increased more. Similarly,the decrease in dosage for subjects exhibiting a ratio above 2.5 mayvary depending on how far above 2.5 the ratio extends. In certainembodiments, measurement of plasma PAA and PAGN ratio takes place afterthe PAA prodrug has had sufficient time to reach steady state (e.g., 48hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2 weeks, or greaterthan 2 weeks after PAA prodrug administration). In certain embodiments,the above steps may be repeated until a desired plasma PAA:PAGN ratio(e.g., 1 to 2.5 or 1 to 2) is achieved. For example, the methods maycomprise measuring plasma PAA and PAGN levels after administration ofthe second dosage, calculating the plasma PAA:PAGN ratio, determiningwhether the PAA prodrug dosage needs to be adjusted based on whether thePAA:PAGN ratio falls within the target range, and administering a thirddosage of the PAA prodrug.

In certain embodiments, methods are provided for treating a nitrogenretention disorder or a condition for which PAA prodrug administrationis expected to be beneficial in a subject that has not previously beenadministered a PAA prodrug. These methods comprise administering a firstdosage of a PAA prodrug, measuring plasma PAA and PAGN levels,calculating the plasma PAA:PAGN ratio, determining whether the PAAprodrug dosage needs to be adjusted based on whether the PAA:PAGN ratiofalls within a target range, and administering a second dosage of thePAA prodrug. In certain embodiments, the target range for PAA:PAGN ratiois 1 to 2.5 or 1 to 2. In certain of these embodiments, the seconddosage is greater than the first dosage if the PAA:PAGN ratio is lessthan 1 (i.e., the dosage is increased) and less than the first dosage ifthe PAA:PAGN ratio is greater than 2.5 (i.e., the dosage is decreased).In other embodiments, the second dosage may or may not be greater thanthe first dosage if the PAA:PAGN ratio is less than 1, depending on oneor more additional characteristics of the subject. In certainembodiments, the second dosage is equal to the first dosage when thePAA:PAGN ratio is 1 to 2.5, i.e., falling within the target range. Incertain embodiments, the target range is divided into one or moresubranges. In certain of these embodiments, the second dosage may beequal to the first dosage if the PAA:PAGN ratio is 1 to 1.5 or 2 to 2.5,but the subject may be subjected to more frequent monitoring. In certainother embodiments, the second dosage may be greater than the firstdosage if the PAA:PAGN ratio is 1 to 1.5 or 1 to 2 and the subject hasrecently exhibited particularly acute symptoms of a nitrogen retentiondisorder or another condition for which PAA prodrug administration isexpected to be beneficial. Similarly, the second dosage may be less thanthe first dosage if the PAA:PAGN ratio is greater than 1.5 or 2 but notgreater than 2.5, depending on the subject's specific clinical orbiochemical characteristics. In certain embodiments, the increase ordecrease in the second dosage versus the first dosage depends on theprecise plasma PAA:PAGN ratio. For example, where the plasma PAA:PAGNratio is only slightly less than 1, the dosage may be increased onlyslightly, but where the PAA:PAGN ratio is significantly less than 1, thedosage may be increased more. Similarly, the decrease in dosage forsubjects exhibiting a ratio above 2.5 may vary depending on how farabove 2.5 the ratio extends. In certain embodiments, measurement ofplasma PAA and PAGN ratio takes place after the PAA prodrug has hadsufficient time to reach steady state (e.g., 48 hours, 48 to 72 hours,72 hours to 1 week, 1 week to 2 weeks, or greater than 2 weeks after PAAprodrug administration). In certain embodiments, the above steps may berepeated until a desired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to2) is achieved. For example, the methods may comprise measuring plasmaPAA and PAGN levels after administration of the second dosage,calculating the plasma PAA:PAGN ratio, determining whether the PAAprodrug dosage needs to be adjusted based on whether the PAA:PAGN ratiofalls within the target range, and administering a third dosage of thePAA prodrug.

A method of administering a PAA prodrug to a subject with a nitrogenretention disorder or another condition for which PAA prodrugadministration is expected to be beneficial. These methods compriseadministering a first dosage of the PAA prodrug, measuring plasma PAAand PAGN levels, calculating the plasma PAA:PAGN ratio, determiningwhether the PAA prodrug dosage needs to be adjusted based on whether thePAA:PAGN ratio falls within a target range, and administering a seconddosage of the PAA prodrug. In certain embodiments, the target range forPAA:PAGN ratio is 1 to 2.5 or 1 to 2. In certain of these embodiments,the second dosage is greater than the first dosage if the PAA:PAGN ratiois less than 1 (i.e., the dosage is increased) and less than the firstdosage if the PAA:PAGN ratio is greater than 2.5 (i.e., the dosage isdecreased). In other embodiments, the second dosage may or may not begreater than the first dosage if the PAA:PAGN ratio is less than 1,depending on one or more additional characteristics of the subject. Incertain embodiments, the second dosage is equal to the first dosage whenthe PAA:PAGN ratio is 1 to 2.5, i.e., falling within the target range.In certain embodiments, the target range is divided into one or moresubranges. In certain of these embodiments, the second dosage may beequal to the first dosage if the PAA:PAGN ratio is 1 to 1.5 or 2 to 2.5,but the subject may be subjected to more frequent monitoring. In certainother embodiments, the second dosage may be greater than the firstdosage if the PAA:PAGN ratio is 1 to 1.5 or 1 to 2 and the subject hasrecently exhibited particularly acute symptoms of a nitrogen retentiondisorder or another condition for which PAA prodrug administration isexpected to be beneficial. Similarly, the second dosage may be less thanthe first dosage if the PAA:PAGN ratio is greater than 1.5 or 2 but notgreater than 2.5, depending on the subject's specific biochemical orclinical characteristics. In certain embodiments, the increase ordecrease in the second dosage versus the first dosage depends on theprecise plasma PAA:PAGN ratio. For example, where the plasma PAA:PAGNratio is only slightly less than 1, the dosage may be increased onlyslightly, but where the PAA:PAGN ratio is significantly less than 1, thedosage may be increased more. Similarly, the decrease in dosage forsubjects exhibiting a ratio above 2.5 may vary depending on how farabove 2.5 the ratio extends. In certain embodiments, measurement ofplasma PAA and PAGN ratio takes place after the PAA prodrug has hadsufficient time to reach steady state (e.g., 48 hours, 48 to 72 hours,72 hours to 1 week, 1 week to 2 weeks, or greater than 2 weeks after PAAprodrug administration). In certain embodiments, the above steps may berepeated until a desired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to2) is achieved. For example, the methods may comprise measuring plasmaPAA and PAGN levels after administration of the second dosage,calculating the plasma PAA:PAGN ratio, determining whether the PAAprodrug dosage needs to be adjusted based on whether the PAA:PAGN ratiofalls within the target range, and administering a third dosage of thePAA prodrug.

In certain embodiments, methods are provided herein for achieving atarget plasma PAA:PAGN ratio in a subject with a nitrogen retentiondisorder or another condition for which PAA prodrug administration isexpected to be beneficial. These methods comprise administering a firstdosage of a PAA prodrug, measuring plasma PAA and PAGN levels,calculating the plasma PAA:PAGN ratio, determining whether the PAAprodrug dosage needs to be adjusted based on whether the PAA:PAGN ratiofalls within a target range, and administering a second dosage of thePAA prodrug based on the PAA:PAGN ratio. If the PAA:PAGN ratio is abovethe target range, the second dosage is less than the first dosage. Ifthe PAA:PAGN ratio is below the target range, the second dosage isgreater than the first dosage. These steps are repeated until a targetplasma PAA:PAGN ratio is achieved. In certain embodiments, the targetratio falls within a target range of 1 to 2.5 or 1 to 2. In certainembodiments, the increase or decrease in the second dosage versus thefirst dosage depends on the precise plasma PAA:PAGN ratio. For example,where the plasma PAA:PAGN ratio is only slightly less than 1, the dosagemay be increased only slightly, but where the PAA:PAGN ratio issignificantly less than 1, the dosage may be increased more. Similarly,the decrease in dosage for subjects exhibiting a ratio above 2.5 mayvary depending on how far above 2.5 the ratio extends. In certainembodiments, measurement of plasma PAA and PAGN ratio takes place afterthe PAA prodrug has had sufficient time to reach steady state (e.g., 48hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2 weeks, or greaterthan 2 weeks after PAA prodrug administration).

In certain embodiments, methods are provided for evaluating the dosageof a PAA prodrug in a subject who has previously been administered afirst dosage of a PAA prodrug. These methods comprise measuring plasmaPAA and PAGN levels, calculating the plasma PAA:PAGN ratio, anddetermining whether the first dosage of the PAA prodrug is effectivebased on whether the PAA:PAGN ratio falls within a target range. Incertain embodiments, the target range for PAA:PAGN ratio is 1 to 2.5 or1 to 2. In certain of these embodiments, the first dosage is consideredtoo low if the PAA:PAGN ratio is less than 1, and too high if thePAA:PAGN ratio is greater than 2.5. In other embodiments, the firstdosage is considered potentially too low if PAA:PAGN ratio is less than1, with a final decision depending on one or more additionalcharacteristics of the subject. In certain embodiments, the target rangeis divided into one or more subranges. In certain of these embodiments,the first dosage is considered potentially effective if the PAA:PAGNratio is 1 to 1.5 or 2 to 2.5, but the subject may be subjected to morefrequent monitoring. In certain other embodiments, the first dosage maybe considered too low if the PAA:PAGN ratio is 1 to 1.5 or 1 to 2 andthe subject has recently exhibited particularly acute symptoms of anitrogen retention disorder or another condition for which PAA prodrugadministration is expected to be beneficial. Similarly, in certainembodiments the first dosage may be considered too high if the PAA:PAGNratio is greater than 1.5 or 2 but not greater than 2.5, depending onthe subject's specific biochemical or clinical characteristics. Incertain embodiments, measurement of plasma PAA and PAGN ratio takesplace after the PAA prodrug has had sufficient time to reach steadystate (e.g., 48 hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2weeks, or greater than 2 weeks after PAA prodrug administration). Incertain embodiments, the methods further comprise a step ofadministering a second dosage that differs from the first dosage, and incertain of these embodiments the above steps may be repeated until adesired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to 2) is achieved.For example, the methods may comprise administering a second dosage thatdiffers from the first dosage, measuring plasma PAA and PAGN levelsafter administration of the second dosage, calculating the plasmaPAA:PAGN ratio, and determining whether the second dosage of the PAAprodrug is effective based on whether the PAA:PAGN ratio falls within atarget range.

In certain embodiments, methods are provided for adjusting the dosage ofa PAA prodrug in a subject who has previously been administered a firstdosage of a PAA prodrug. These methods comprise measuring plasma PAA andPAGN levels, calculating the plasma PAA:PAGN ratio, and determiningwhether to adjust the dosage of the PAA prodrug based on whether thePAA:PAGN ratio falls within a target range. In certain embodiments, thetarget range for PAA:PAGN ratio is 1 to 2.5 or 1 to 2. In certain ofthese embodiments where the target range is 1 to 2.5, a PAA:PAGN ratioof less than 1 indicates the PAA prodrug dosage needs to be adjustedupwards, while a PAA:PAGN ratio above 2.5 indicates the PAA prodrugdosage needs to be adjusted downwards. In other embodiments, a PAA:PAGNratio of less than 1 indicates that the PAA prodrug dosage potentiallyneeds to be adjusted upwards, with a final decision depending on one ormore additional characteristics of the subject. In certain embodiments,the target range is divided into one or more subranges. In certain ofthese embodiments, a PAA:PAGN ratio of 1 to 1.5 or 2 to 2.5 indicatesthat the dosage need not be adjusted, but that the subject should besubjected to more frequent monitoring. In certain other embodiments, aPAA:PAGN ratio of 1 to 1.5 or 1 to 2 indicates that the dosage needs tobe increased when the subject has recently exhibited particularly acutesymptoms of a nitrogen retention disorder or another condition for whichPAA prodrug administration is expected to be beneficial. Similarly, incertain embodiments a PAA:PAGN ratio greater than 1.5 or 2 but notgreater than 2.5 may indicate that the dosage needs to be decreased,depending on the subject's specific biochemical or clinicalcharacteristics. In certain embodiments, measurement of plasma PAA andPAGN ratio takes place after the PAA prodrug has had sufficient time toreach steady state (e.g., 48 hours, 48 to 72 hours, 72 hours to 1 week,1 week to 2 weeks, or greater than 2 weeks after PAA prodrugadministration). In certain embodiments where a determination is madethat the dosage needs to be adjusted, the methods further comprise astep of administering a second dosage that differs from the firstdosage, and in certain of these embodiments the above steps may berepeated until a desired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to2) is achieved. For example, the methods may comprise administering asecond dosage that differs from the first dosage, measuring plasma PAAand PAGN levels after administration of the second dosage, calculatingthe plasma PAA:PAGN ratio, and determining whether the second dosage ofthe PAA prodrug needs to be adjusted based on whether the PAA:PAGN ratiofalls within a target range. In certain embodiments, the increase ordecrease in the second dosage versus the first dosage depends on theprecise plasma PAA:PAGN ratio. For example, where the plasma PAA:PAGNratio is only slightly less than 1, the dosage may be increased onlyslightly, but where the PAA:PAGN ratio is significantly less than 1, thedosage may be increased more. Similarly, the decrease in dosage forsubjects exhibiting a ratio above 2.5 may vary depending on how farabove 2.5 the ratio extends.

In certain embodiments, methods are provided for optimizing thetherapeutic efficacy of a PAA prodrug for use in treating a nitrogenretention disorder in a subject. These methods comprise measuring plasmaPAA and PAGN levels in a subject who has previously been administered aPAA prodrug, calculating the plasma PAA:PAGN ratio, determining whetherto adjust the dosage of the PAA prodrug based on whether the PAA:PAGNratio falls within a target range, and administering an adjusted dosageof the PAA prodrug as necessary. These steps are repeated until thesubject exhibits a plasma PAA:PAGN ratio falling within the target range(e.g., 1 to 2.5 or 1 to 2). In certain embodiments where the targetrange is 1 to 2.5, a plasma PAA:PAGN ratio of less than 1 indicates thatthe dosage needs to be adjusted upwards, while a ratio greater than 2.5indicates that the dosage needs to be decreased. In certain embodiments,the target range is divided into one or more subranges. In certain ofthese embodiments, a PAA:PAGN ratio of 1 to 1.5 or 2 to 2.5 indicatesthat the dosage does not need to be adjusted, but that the subjectshould be subjected to more frequent monitoring. In certain otherembodiments, a PAA:PAGN ratio of 1 to 1.5 or 1 to 2 indicates that thedosage needs to be increased when the subject has recently exhibitedparticularly acute symptoms of a nitrogen retention disorder or anothercondition for which PAA prodrug administration is expected to bebeneficial. Similarly, in certain embodiments a PAA:PAGN ratio greaterthan 1.5 or 2 but not greater than 2.5 may indicate that the dosageneeds to be decreased, depending on the subject's specific biochemicalor clinical characteristics. In certain embodiments, measurement ofplasma PAA and PAGN ratio takes place after the PAA prodrug has hadsufficient time to reach steady state (e.g., 48 hours, 48 to 72 hours,72 hours to 1 week, 1 week to 2 weeks, or greater than 2 weeks after PAAprodrug administration). In certain embodiments, the magnitude of theincrease or decrease in dosage may be based on the precise PAA:PAGNratio. For example, a PAA:PAGN ratio that is slightly less than 1 mayindicate that the dosage needs to be increased slightly, while a ratiosignificantly less than 1 may indicate the dosage needs to be increasedto a greater degree. In certain embodiments, the above steps arerepeated until the subject exhibits a PAA:PAGN ratio falling within thetarget range.

In certain embodiments, methods are provided for determining whether aprescribed first dosage of a PAA prodrug can be safely administered to asubject. These methods comprise administering the prescribed firstdosage to the subject, measuring plasma PAA and PAGN levels, calculatingthe plasma PAA:PAGN ratio, and determining whether the prescribed firstdosage is safe for the subject based on whether the PAA:PAGN ratio fallsabove a target range, wherein a PAA:PAGN ratio falling above the targetrange indicates that the first dosage cannot be or potentially cannot besafely administered to the subject. In certain embodiments, the targetrange for PAA:PAGN ratio is 1 to 2.5 or 1 to 2. In certain of theseembodiments where the target range is 1 to 2.5, a PAA:PAGN ratio above2.5 indicates the PAA prodrug dosage is unsafe and needs to be adjusteddownwards. In certain embodiments, the target range is divided into oneor more subranges. In certain of these embodiments, a PAA:PAGN ratio of2 to 2.5 indicates that the first dosage is safe, but that the subjectshould be subjected to more frequent monitoring. In other embodiments, aPAA:PAGN ratio of 2 to 2.5 indicates that the first dosage ispotentially unsafe, with a final determination of safety taking intoaccount the subject's specific biochemical or clinical characteristics.In certain embodiments, measurement of plasma PAA and PAGN ratio takesplace after the PAA prodrug has had sufficient time to reach steadystate (e.g., 48 hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2weeks, or greater than 2 weeks after PAA prodrug administration). Incertain embodiments where a determination is made that the first dosageis unsafe and needs to be decreased, the methods further comprise a stepof administering a second dosage that is lower than the first dosage,and in certain of these embodiments the above steps may be repeateduntil a desired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to 2) isachieved. For example, the methods may comprise administering a seconddosage that is lower than the first dosage, measuring plasma PAA andPAGN levels after administration of the second dosage, calculating theplasma PAA:PAGN ratio, and determining whether the second dosage of thePAA prodrug can be safely administered to the subject based on whetherthe PAA:PAGN ratio falls above a target range.

In certain embodiments, methods are provided for determining whether aprescribed first dosage of a PAA prodrug will be effective for treatinga nitrogen retention disorder or another disorder for which PAA prodrugadministration is expected to be beneficial. These methods compriseadministering the prescribed first dosage to the subject, measuringplasma PAA and PAGN levels, calculating the plasma PAA:PAGN ratio, anddetermining whether the prescribed first dosage will be effective forthe subject based on whether the PAA:PAGN ratio falls below a targetrange, wherein a PAA:PAGN ratio falling below the target range indicatesthat the first dosage will not be or potentially will not be effectivefor treating a disorder. In certain embodiments, the target range forPAA:PAGN ratio is 1 to 2.5 or 1 to 2. In certain of these embodimentswhere the target range is 1 to 2.5, a PAA:PAGN ratio below 1 indicatesthe PAA prodrug dosage is unlikely to be effective and needs to beadjusted upwards. In other embodiments, a PAA:PAGN ratio below 1indicates that the first dosage is potentially ineffective, with a finaldetermination of whether the dosage is likely to be ineffective based onthe subject's specific biochemical or clinical characteristics. Incertain embodiments, the target range is divided into one or moresubranges. In certain of these embodiments, a PAA:PAGN ratio of 1 to 1.5indicates that the first dosage is likely to be effective, but that thesubject should be subjected to more frequent monitoring. In otherembodiments, a PAA:PAGN ratio of 1 to 1.5 indicates that the firstdosage is potentially ineffective, with a final determination of whetherthe dosage is likely to be ineffective taking into account the subject'sspecific biochemical or clinical characteristics. In certainembodiments, measurement of plasma PAA and PAGN ratio takes place afterthe PAA prodrug has had sufficient time to reach steady state (e.g., 48hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2 weeks, or greaterthan 2 weeks after PAA prodrug administration). In certain embodimentswhere a determination is made that the first dosage is likely to beineffective and needs to be increased, the methods further comprise astep of administering a second dosage that is higher than the firstdosage, and in certain of these embodiments the above steps may berepeated until a desired plasma PAA:PAGN ratio (e.g., 1 to 2.5 or 1 to2) is achieved. For example, the methods may comprise administering asecond dosage that is higher than the first dosage, measuring plasma PAAand PAGN levels after administration of the second dosage, calculatingthe plasma PAA:PAGN ratio, and determining whether the second dosage ofthe PAA prodrug is likely to be ineffective for treating a disorderbased on whether the PAA:PAGN ratio falls above a target range.

Provided herein in certain embodiments are methods for monitoringtherapy with a PAA prodrug in patients with a nitrogen retentiondisorder. These methods comprise administering a PAA prodrug to thesubject, measuring plasma PAA and PAGN levels, and calculating theplasma PAA:PAGN ratio. In these methods, a PAA:PAGN ratio falling withina target range (e.g., 1 to 2.5 or 1 to 2) indicates that the therapy iseffective, while a ratio falling outside this range indicates that thetherapy may need to be adjusted. In certain embodiments, the plasmaPAA:PAGN ratio is compared to a previously obtained PAA:PAGN ratio fromthe same subject to evaluate the effectiveness of PAA prodrugadministration.

In certain embodiments, the methods provided herein may be used inconjunction with the methods described in WO09/134460 and WO10/025303.In these embodiments, urinary PAGN levels may be determined in additionto plasma PAA:PAGN ratio, with both measurements being used to evaluateor adjust PAA prodrug dosage.

A “PAA prodrug” as used herein refers to any drug that contains or isconverted to PAA following administration to a subject, or to anypharmaceutically acceptable salt, ester, acid, or derivative thereof. APAA prodrug may be administered via any route, including oral orparenteral administration. A PAA prodrug may be converted directly toPAA (e.g., a salt or ester of PAA; PBA or a salt or ester thereof suchas NaPBA), or it may be converted to PAA via an intermediate (e.g., apre-prodrug such as HPN-100). Other examples of PAA prodrugs includebutyroyloxymethyl-4-phenylbutyrate.

An adjustment to the dosage of a PAA prodrug as discussed herein mayrefer to a change in the amount of drug per administration (e.g., anincrease from a first dosage of 3 mL to a second dosage of 6 mL), achange in the number of administration within a particular time period(e.g., an increase from once a day to twice a day), or any combinationthereof.

A “subject in need thereof” as used herein refers to any individualhaving a condition or suspected of having a condition for whichadministration of a PAA prodrug is expected to be beneficial. Forexample, a subject may be an individual with a nitrogen retentiondisorder or suspected of having a nitrogen retention disorder, includingfor example UCD, HE, and/or kidney failure/ESRD (Lee 2010; McGuire 2010;Lichter 2011). Likewise, a subject may have or be suspected of havinganother condition for which PAA prodrug administration is expected to bebeneficial, including for example cancer (Thiebault 1994; Thiebault1995), neurodegenerative disorders such as Huntington's Disease (Hogarth2007), amyotrophic lateral sclerosis (ALS) (Cudkowicz 2009), and spinalmuscular atrophy (SMA) (Mercuri 2004; Brahe 2005), metabolic disorders(e.g., maple syrup urine disease (MSUD) (Bruneti-Pieri 2011), or sicklecell disease (Hines 2008).

A subject that has previously been administered a PAA prodrug may havebeen administered the drug for any duration of time sufficient to reachsteady state. For example, the subject may have been administered thedrug over a period of 2 to 7 days, 1 week to 2 weeks, 2 weeks to 4weeks, 4 weeks to 8 weeks, 8 weeks to 16 weeks, or longer than 16 weeks.

A “PAA prodrug” as used herein refers to any drug that contains or isconverted to PAA following administration to a subject, or to anypharmaceutically acceptable salt, ester, acid, or derivative thereof. APAA prodrug may be administered via any route, including oral orparenteral administration. A PAA prodrug may be converted directly toPAA (e.g., PBA or a salt thereof such as NaPBA), or it may be convertedto PAA via an intermediate (e.g., a pre-prodrug such as HPN-100). Otherexamples of PAA prodrugs include butyroyloxymethyl-4-phenylbutyrate.

An adjustment to the dosage of a PAA prodrug as discussed herein mayrefer to a change in the amount of drug per administration (e.g., anincrease from a first dosage of 3 mL to a second dosage of 6 mL), achange in the number of administration within a particular time period(e.g., an increase from once a day to twice a day), or any combinationthereof.

The terms “treat,” “treating,” or “treatment” as used herein may referto preventing a disorder, slowing the onset or rate of development of adisorder, reducing the risk of developing a disorder, preventing ordelaying the development of symptoms associated with a disorder,reducing or ending symptoms associated with a disorder, generating acomplete or partial regression of a disorder, or some combinationthereof. For example, where the disorder being treated is a nitrogenretention disorder, “treating” may refer to lowering waste nitrogenlevels below a threshold level, preventing waste nitrogen levels fromreaching a threshold level, decreasing the likelihood of waste nitrogenlevels exceeding a threshold level, reducing or ending symptomsassociated with elevated waste nitrogen levels, or a combinationthereof.

With regard to the methods of treatment disclosed herein, interpretationof the PAA:PAGN ratio must be performed in the context of thetherapeutic objective. For example, in subjects being treated for anitrogen retention disorder, the therapeutic objective is elimination ofwaste nitrogen in the form of PAGN. In subjects being treated for otherdisorders for which PAA prodrug administration is expected to bebeneficial (e.g., neurodegenerative disorders, MSUD), the therapeuticobjective is safely achieving target plasma levels of PAA and/or PBA.

Any methods known in the art may be used to obtain a plasma bloodsample. For example, blood from a subject may be drawn into a tubecontaining heparin or ethylenediaminetetraacetic acid (EDTA). In certainembodiments, the sample can be placed on ice and centrifuged to obtainplasma within 15 minutes of collection, stored at 2-8° C. (36-46° F.)and analyzed within 3 hours of collection. In other embodiments, theblood plasma sample is snap frozen, stored at ≦−18° C. (≦0° F.) andanalyzed at a later time. For example, the sample may be analyzed at0-12 hours, 12-24 hours, 24-48, 48-96 hours after freezing, or withinany other timeframe over which the sample has demonstrated stability. Incertain of these embodiments, the blood sample is stored at atemperature between 0-15° C., such as 2-8° C. In other embodiments, theblood sample is stored below 0° C. or below −18° C.

Measurement of PAA and PAGN levels in a plasma sample is carried outusing techniques known in the art. For example, PAA and PAGN levels maybe measured using liquid chromatography/mass spec analyses.

Any combination of embodiments described herein can be envisioned.Although individual features may be included in different claims, thesemay be advantageously combined.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1 Analysis of PAA:PAGN Ratio in UCD and HE Subjects

Plasma PAA and PAGN levels and PAA:PAGN ratio were analyzed in more than4000 plasma samples obtained from various clinical trials of healthyadults, severely hepatic impaired adults with clinically decompensatedChild-Pugh B or C cirrhosis, and UCD patients ages 29 days or older.Healthy and hepatically impaired adults received HPN-100, while UCDsubjects received both HPN-100 and NaPBA. Clinical trial populations aresummarized in Tables 1 and 2.

TABLE 1 Clinical studies and analysis populations Study ProtocolsAnalysis Group Description Demographics Included Populations 1Short-term (<=2-4 weeks) Adults and children UP 1204-003 A, B exposurein UCD subjects ages 29 days or HPN-100-005SO greater (N = 81)HPN-100-006 HPN-100-012 2 Long-term exposure in Adults and childrenHPN-100-005SE A UCD and HE subjects ages 6 years or HPN-100-007 greater(N = 180) HPN-100-008 Part B 3 Short-term (<=4 weeks) Adults (N = 15)HPN-100-008 Part A A, B exposure in hepatic impaired subjects 4Short-term exposure (<=4 Adults (N = 98) HPN-100-010 A, B weeks) inhealthy subjects

TABLE 2 Demographics and number of samples used No. of No. oftime-specific No. of sample points PK sample points subjects (PopulationA) (Population B) Attribute Count Percent Count Percent Count PercentPopulation Healthy 86 17.0 2126 34.4 2126 38.5 Hepatic 103 20.4 830 13.4830 15.0 Encephalopathy (HE) UCD 158 31.3 1616 26.1 1281 23.2 Total 347100.0 4572 100.0 4237 100.0 Age 29 days-<6 yrs 15 4.3 110 2.4 110 2.66-<18 yrs 47 13.5 373 8.2 213 5.0 18+ yrs 285 82.1 4089 89.4 3914 92.4Sex F 199 57.3 2394 52.4 2152 50.8 M 148 42.7 2178 47.6 2085 49.2

Analysis Population A consisted of quantifiable levels of PAA and PAGNmetabolites derived from all studies described above. All PAA and PAGNlevels used for analysis came from blood samples drawn once dosing withNaPBA or HPN-100 had reached steady state. Analysis Population Bconsisted of quantifiable levels of PAA and PAGN metabolites duringstudies in which pharmacokinetics were analyzed and for which blooddraws were performed over 12 or 24 hours at steady state and for whichthe timing of the blood sample in relation to dosing was known. Subjectsin study groups 1, 3 and 4 above contributed to these points. AnalysisPopulation B was the source of analyses that examined how PAA levelschanged with time relative to dosing, where dosing could have been witheither NaPBA or HPN-100. To be eligible for Analysis Population B, thetime of the blood draw relative to the time of initiation of dosingduring the dosing period had to have been recorded.

Data on metabolite levels were pooled across a wide range of agelevels—infants, toddlers, children, adolescents, and adults. Allchildren, defined as ages under 18, were UCD patients. The majority ofthe blood sampling points came from adults (89.4%). Newborn infants (<29days old) were not studied in any of the clinical trials for theinvestigational agent HPN-100. The population of blood sampling pointswere roughly equally divided between female and male (57.3% female,42.7% male).

To examine the predictive ability of PAA:PAGN ratios, a subject wasconsidered to have achieved a high value of PAA if any PAA value up to24 hours since initiation of dosing equaled or exceeded 400 μg/mL orequaled or exceeded 500 μg/mL. PAA:PAGN ratios were grouped into one ofthree categorization schemes: a.) [0−<=2.0], [>2.0], b.)[0−<=2.5, >2.5], c.) [0−<=3.0, >3.0]. The repeated measures categoricaloutcome was modeled using GEE with a logit link function, ratio categoryas the independent variable, and SUBJECTID as the repeated measuresfactor. Confidence intervals for the predicted probabilities werecomputed by bootstrap estimation of 1000 resamplings of the originaldata, as detailed in Davison & Hinkley, “Bootstrap Methods and TheirApplication,” Cambridge Univ. Press (1997), pp. 358-362.

Results are summarized in FIGS. 2-5. A striking curvilinear relationshipwas observed between plasma PAA levels and PAA:PAGN ratio at any giventimepoint. FIG. 2A shows the relationship between the ratio of PAA:PAGNconcentrations and absolute PAA levels in micrograms per milliliteramong blood samples that had quantifiable values for both PAA and PAGN.The ratio axis (i.e. ‘X’ axis) is plotted on a logarithmic (base e)scale. For ratios less than 1.0, increases in ratio are not associatedwith correspondingly elevated or increased levels of PAA. Above ratiosof 1.0, there is a gradual increase in PAA levels, and a noticeableupswing in PAA levels that begins in the vicinity of a ratio of 2.0.This finding suggests that when the ratio of PAA precursor to PAGNproduct approaches higher values, the values of PAA are alsocorrespondingly high. This increase in the ratio of precursor (PAA) toproduct (PAGN) implies ineffective PAA to PAGN conversion, regardless ofwhether the PAA is derived from HPN-100 or NaPBA.

To determine whether excessive PAA build-up is a function of dosing, theplots mentioned above were repeated, but this time adjusting forassigned dose level of NaPBA or HPN-100 at the time of the blood draw.Since the UCD population consisted of a mixture of children and adultsundergoing both short-term therapy and long-term therapy, total assigneddaily dose for UCD patients was standardized to body surface area andreported in PBA-equivalent grams meter². Healthy and HE subjects wereall adults and their assigned dose was not adjusted by body surfacearea. Dose levels for healthy and HE subjects were reported in HPN-100equivalent mL. Dose levels for UCD subjects were reported inNaPBA-equivalent grams.

The excess of PAA over PAGN, indicated by larger ratios as PAAincreases, was evident across all dosage groups, disease populations,and types of treatment in UCD patients (i.e., applies to both NaPBA andHPN-100). This finding suggests that analysis of the precursor (PAA) toproduct (PAGN) ratio may be predictive of the efficiency of conversionamong patients with or without liver dysfunction (UCD patients havenormal liver function apart from their urea cycle dysfunction) andindependently of dose. As a corollary, the presence of liver dysfunction(e.g. cirrhosis) by itself, is not necessarily a reliable determinant ofwhether a particular patient is at risk for high PAA levels.

The ability of PAA:PAGN ratios to predict extremely high plasma PAAconcentrations was determined by modeling the probability that a subjectwould exceed a PAA value of 400 or 500 μg/mL anytime during a 24 hourdosing period, based on the ratio of PAA to PAGN computed at pre-dose(presumably trough), 12 hours after dosing (presumably peak), and themaximum ratio encountered anytime between pre-dose and 12 hourspost-dose. This interval of 0-12 hours was chosen for practical reasons,as it would encompass the entire interval corresponding to the usualoutpatient visit.

Since subjects could have multiple dosing periods within a givenclinical study, the probability was modeled using Generalized EstimatingEquations. Three categorizations of ratios were modeled: a.) [0−<=2.0][>2.0], b.) [0−<=2.5, >2.5], c.) [0−<=3.0, >3.0]. The models wererepeated with PAA values greater than or equal to 500 μg/mL consideredextreme. Results are summarized in Table 3.

TABLE 3 Probabilities of extreme PAA values encountered during 24 hourPK sampling with PAA:PAGN ratios (all subjects combined) Probabilitythat Time of a Subject With Bootstrapped Blood Draw Observed This RatioWill 95% PAA Value Considered Used For Ratio Ratio of Exceed HighConfidence High Classification PAA/PAGN Value* (%) Interval**[<=2.0, >2.0] >=400 μg/mL t = 0 (fasting) <=2.0 0.005 (0.5%)  0.004,0.020 >2.0 0.164 (16.4%) 0.041, 0.281 t = 12 hours <=2.0 0.003 (0.3%) 0.004, 0.021 >2.0 0.227 (22.7%) 0.048, 0.412 MAX(0-12) <=2.0 0.002(0.2%)  0.004, 0.010 >2.0 0.143 (14.3%) 0.036, 0.263 >=500 μg/mL t = 0(fasting) <=2.0 did not converge >2.0 t = 12 hours <=2.0 did notconverge >2.0 MAX(0-12) <=2.0 did not converge >2.0 [<=2.5, >2.5] >=400μg/mL t = 0 (fasting) <=2.5 0.008 (0.8%)  0.004, 0.023 >2.5 0.191(19.1%) 0.053, 0.366 t = 12 hours <=2.5 0.007 (0.7%)  0.004, 0.016 >2.50.364 (36.4%) 0.125, 0.752 MAX(0-12) <=2.5 0.003 (0.3%)  0.004,0.013 >2.5 0.200 (20.0%) 0.050, 0.381 >=500 μg/mL t = 0 (fasting) <=2.50.003 (0.3%)  0.004, 0.011 <=2.5 0.084 (8.4%)  0.029, 0.214 t = 12hours >2.5 did not converge >2.5 MAX(0-12) <=2.5 did not converge >2.5[<=3, >3] >=400 μg/mL t = 0 (fasting) <=3.0 0.010 (1.0%)  0.004,0.025 >3.0 0.205 (20.5%) 0.059, 0.398 t = 12 hours <=3.0 0.013 (1.3%) 0.004, 0.028 >3.0 0.250 (25.0%) 0.113, 0.576 MAX(0-12) <=3.0 0.003(0.3%)  0.004, 0.014 >3.0 0.229 (22.9%) 0.059, 0.438 >=500 μg/mL t = 0(fasting) <=3.0 0.003 (0.3%)  0.004, 0.010 >3.0 0.102 (10.2%) 0.032,0.255 t = 12 hours <=3.0 did not converge >3.0 MAX(0-12) <=3.0 did notconverge >3.0 Analysis repeated for each ratio cut off categoryindependently. *Probability derived from Generalized EstimatingEquations model with logit link function. **Confidence interval derivedfrom method disclosed in Davison & Hinkley, “Bootstrap Methods and TheirApplication,” Cambridge Univ. Press (1997), pp. 358-362, using 1000re-samplings of original data.

Because of the sparseness of samples in which PAA equaled or exceeded500 μg/mL, 400 μg/mL proved to be a more stable and predictable target(i.e. high) value. Of the three categorizations of ratio considered, thecutpoint of 2.5 was the best discriminator and predictor of the risk ofexperiencing an high value. For example, referring to Table 3, a subjectwith a PAA:PAGN ratio >2.5 at t=12 hours after dosing has a 36.4% chance(95% c. i.=0.125, 0.752) of exceeding 400 μg/mL in PAA sometime duringthe 24-hour PK sampling period.

Results were similar whether the ratio was computed from plasma drawn atpre-dose, 12 hours after initiation of dosing, or the maximum ratioencountered anytime between pre-dose and 12 hours after initiation ofdosing.

Due to the very high intra-day variability of plasma PAA levels, aPAA:PAGN ratio observed as exceeding 2.0 at a certain time followingdosing may not remain greater than 2.0 in subsequent times. To evaluatethe optimal time for obtaining a PAA:PAGN ratio measurement (i.e., thetime that gives the greatest probability of correctly detecting asubject whose PAA:PAGN ratio ever equals or exceeds 2.0 during thedosing period), ratios were evaluated at 0 (pre-dose) and 2, 4, 6, 8,10, and 12 hours post-dosing and modeled using GEE methodology. Pairwisedifferences in sensitivity between time points were evaluated using LSmeans and confidence intervals were computed.

FIG. 3 plots the estimated probabilities of correctly detecting a ratioprofile that ever equals of exceeds 2.0. With the exception of time=2hours and time=10 hours, time points of 0, 4, 6, 8, and 12 hourspost-dosing were equally effective in detecting subjects who equal orexceed a PAA:PAGN ratio of 2.0 at some point during the dosing period.Sensitivities were in the range of 75-90 percent. There were too fewblood samples collected at t=10 hours to analyze inter-time differences.Differences in predictive value were observed. For example, bloodsamples collected at t=2 hours post-dosing had a significantly lowerprobability of detecting subjects who equal or exceed a PAA:PAGN ratioof 2.0 than samples collected at t=0 (p=0.036), 4 (p=0.032), or 6 hours(p=0.017) post-dosing (p values are comparisons of t=2 hour probabilitywith other time points). Similarly, a sample collected at t=12 hoursfollowing initiation of dosing had the highest probability (87%) ofdetecting a subject whose ratio ever equals or exceeds 2.0. However, forpractical clinical purposes, the differences in predictive value amongtime points was trivial relative to the dramatically greater variabilityin PAA values themselves, meaning that random blood draws can be usedfor measurement of PAA:PAGN ratio.

Further exploration of the fluctuation of PAA:PAGN ratios over time wasconducted by dividing the subject population into cohorts according tothe maximum PAA:PAGN ratio achieved during the 24-hour PK sampling timeduring the dosing period. Cohorts were divided into “low” (maximum ratio<=2.0), “medium” (maximum ratio: 2.01-2.50), and “high” (maximumratio >2.50). Each cohort was then followed over time during the dosingperiod at t=0 hours(pre-dose), 4, 6, and 8 hours post-dosing and thedistribution of PAA:PAGN ratios within the cohort summarized using abox-and-whisker plot at each time point. This analysis was conducted forthe PK-timepoint-specific population as a whole (analysis population B)as well as for each disease subpopulation separately.

FIG. 4 plots the progression of ratios for all subjects combined. Each“panel” of the plot that divides the graphing space into thirdsrepresents one cohort. Subjects in the high cohort had high ratiosthroughout the day and not only at a particular time point. Therefore,subjects in this cohort (n=73 subject/dosing periods) started with highratios (median ratio >2.5) and remained high throughout the first 12hours. This finding is consistent with the findings plotted in FIG. 3which revealed the consistency of sensitivity in ratios.

The relationship between PAA levels and PAA:PAGN ratios was furtheranalyzed by categorizing ratios into “low” (maximum ratio <=2.0),“medium” (maximum ratio: 2.01-2.50), and “high” (maximum ratio >2.50).Unlike the previous analysis, this analysis did not associatesubject/dosing periods with particular cohorts (i.e., all samples andall time points are combined with regard to the subject or dosingperiod).

FIG. 5A shows the box-and-whisker plots of PAA levels grouped by theabove categories of PAA:PAGN ratio for all subjects, while FIG. 5B showsthe same for UCD and HE subjects only. The results were very similar inboth analysis sets. Following a statistically significant overallKruskal-Wallis test (p<0.0001), pairwise comparisons of PAA levels wereconducted using Wilcoxon-Mann-Whitney with a Bonferroni alpha correctionof (0.0167). In both analysis sets, ratios greater than 2.5 hadsignificantly higher PAA levels (p<0.001) than either ratios between2.0-2.5 or ratios less than 2.0. Furthermore, ratios between 2.0-2.5were associated with significantly higher PAA levels than ratios lessthan 2.0 (p<0.001).

Example 2 Analysis of PAA:PAGN Ratio as a Guide to Dose Adjustment andMonitoring in a UCD Patient

Patient 1 was a 15 year old partial OTC female receiving HPN-100 asmaintenance therapy for her UCD at a dose of 9 mL/day. The patient'sammonia had been controlled since her last routine visit around 6 monthsago, but she was complaining of headache and lack of appetite for thepast 3 days. Ammonia and metabolite levels were tested after overnightfasting and showed the following results: ammonia 55 μmol/L, PAA andPAGN below levels of quantification. The physician suspectednon-compliance with drug and repeated the tests in midday several hoursafter lunch and found the following results: ammonia: 117 μmol/L; PAA 55μg/L, PAGN 121 μg/L, and PAA:PAGN ratio approximately 0.5. The patientindicated that she had been fully compliant with her medication. Basedon the PAA to PAGN ratio of 0.5 and ammonia of 117, the physiciandecided to increase the dosage of HPN-100 to 12 mL/day. After one weekof treatment with the new dose of HPN-100, all symptoms resolved and thelaboratory tests after overnight fasting showed the following: ammonia 9μmol/L; PAA 12.9 μg/L, PAGN of 9 μg/L, and PAA:PAGN ratio of 1.3. Middaytests showed the following: ammonia 35 μmol/L, PAA 165 μg/L, PAGN 130μg/L, and PAA:PAGN ratio of ˜1.2. The patient was considered controlledand the dose remained at 12 mL/day.

Example 3 Analysis of PAA:PAGN Ratio as a Guide to Dose Adjustment in aUCD Patient

Patient 2 was a 1 year old male OTC receiving 600 mg/kg of NaPBA perday. The patient presented with poor feeding and somnolence. Laboratorytests showed ammonia levels of <9 μmol/L, PAA levels of 530 μg/L, PAGNlevels of 178 μg/L, and a PAA:PAGN ratio of >2.5, suggesting that thedose of NaPBA was greater than the patient could effectively convert toPAGN. The treating physician decided to decrease the dose of NaPBA to450 mg/Kg/day. After one week of treatment with the new dosage, thepatient's mother reported that he was eating well and was no longersomnolent. Laboratory tests showed the following: ammonia 20 μmol/L, PAA280 μg/L, and PAGN 150 μg/L.

Example 4 Analysis of PAA:PAGN Ratio as a Guide to Assessment ofImportance of a High PAA Level in a UCD Patient

Patient 3 is a 25 year old OTC female who is being treated with HPN-100.The physician had to increase the dose of HPN-100 several times in orderto achieve clinical and blood ammonia within normal limits. Patient 3was treated at a dose of 18 mL/day for her UCD for the past month. Inher next office visit, she did not have any complaints and the followinglab results were reported: ammonia 22 μmol/L, PAA 409 μg/L, PAGN 259μg/L, and PAA:PAGN ratio of 1.5. Despite the patient's relatively highPAA levels, the PAA:PAGN ratio indicated that the subject was beingadequately treated and that the patient was able to effectivelymetabolize the high dose of HPN-100 that she was receiving. Thephysician decided to continue the treatment as planned.

Example 5 Analysis of PAA:PAGN Ratio as a Guide to Dose Adjustment in aPatient With Spinal Muscular Atrophy and Concomitant Liver Disease

Patient 4 was a 2 year old female being treated with a liquid form ofNaPBA for her type II SMA. The patient also suffered from chronichepatitis C virus infection acquired perinatally from her infectedmother. The patient had been having mild to moderate elevation oftransaminases since birth, with episodes of icterus and a recent liverbiopsy has confirmed presence of chronic hepatitis and cirrhosis. Thepatient was receiving 4 g of NaPBA per day, and the physician wanted toincrease the dosage due to the patient's growth but was concerned aboutthe effects of liver dysfunction on drug metabolism. The physicianordered plasma PAA and PAGN levels and the results were as follows: PAA110 μg/L, PAGN 85 μg/L, PAA:PAGN ratio of 1.2. The physician decided toincrease the dosage of NaPBA to 6 g/day, and repeated the plasmametabolite level measurements after one week of treatment with the newregimen. The results were as follows: PAA 155 μg/L, PAGN 110 μg/L, andPAA:PAGN ratio of 1.4. The physician decided to leave the patient on 6g/day of NaPBA since his liver seems to have adequate capacity tometabolize 6 g of NaPBA.

Example 6 Analysis of PAA:PAGN Ratio as a Guide to Dose Adjustment in aPatient With Huntington's Disease and Concomitant Liver Disease

Patient 5 was a 56 year old male diagnosed with Huntington's diseaseseveral years ago. He also had a history of alcohol abuse and wasdiagnosed with alcoholic cirrhosis last year. His wife enrolled him inclinical trials that involved an experimental drug delivering PBA at aslow rate, thereby enabling once-a-day dosing of the drug. The study hadan option for dose escalation after 2 weeks of treatment if clinicallysafe. Although the protocol did not exclude patients with liverdysfunction, the investigator was concerned about PBA metabolism andpossible accumulation of PAA in higher doses due to the patient's liverdysfunction. The investigator enrolled the patient in the low dose groupand performed plasma PBA, PAA and PAGN measurements after 6 weeks oftreatment with experimental drug. The patient reported improvement inhis HD symptoms with no specific complains. Plasma metabolite levelsafter six weeks of treatment were as follows: PBA 45 μg/L; PAA 159 μg/L,and PAGN 134 μg/L. The dosage of the drug was increased by 50%. Afterfour days of treatment at the new dosage, the patient started tocomplain about short episodes of somnolence. The investigator performeda blood test and observed the following: PBA 44 μg/L; PAA 550 μg/L, PAGN180 μg/L, and PAA:PAGN ratio of >3. The PAA:PAGN ratio of greater than2.5 indicated that the patient's liver could not effectively metabolizethe higher dose of the drug, and the investigator therefore decided toreduce the dosage of the experimental drug and not continue doseescalation.

Example 7 Analysis of PAA:PAGN Ratio as a Guide to Dose Adjustment in aPatient With MSUD

Patient 6 was a 4 year old female being treated with HPN-100 for MSUD.The patient was receiving 6 mL of HPN-100 once a day, and the physicianwanted to increase the dosage due to the patient's growth. Midday plasmaPAA and PAGN measurements after the dose of medication were as follows:PAA 550 μg/L, PAGN 180 μg/L, and PAA:PAGN ratio of >2.5. The physicianbelieved a lower dosage of HPN-100 would not be as effective for thepatient, and decided to change the dosing regimen to 3 mL BID instead of6 mL QD based on the high PAA:PAGN ratio. The tests were repeated afterone week of treatment with the new BID regimen, with the followingresults: PAA 350 μg/L, PAGN 190 μg/L, and PAA:PAGN ratio of 1.8. Basedon the ratio of 1.8, the physician decided to leave the patient on 3 mLBID since she can efficiently use a total dose of 6 mL/day given individed doses but not as a bolus.

Example 8 Analysis of PAA:PAGN Ratio as a Guide to Monitor a PatientWith HE and Hepatic Impairment

Patient 7 was a 55 year old Caucasian male diagnosed with alcoholiccirrhosis 3 years ago. His transaminase levels had been mildly elevatedand he had recently experienced mild episodes of HE. In the lastassessment at the time of hospital admission for a grade 2 HE episode,the patient had a blood ammonia of 85 μmol/L, ALT of 55 U/L, and AST of47 U/L, and a calculated MELD score of 11. The physician decided tostart an ammonia scavenging therapy for the patient and treated him withHPN-100 6 mL BID. The patient returned for a follow up visit after 3months, during which time he had experienced no episodes of HE. Hislaboratory assessments showed the following: ammonia of 30 μmol/L,plasma PAA level of 285 μg/mL, PAGN level of 120 μg/L, ALT of 66 U/L,AST of 50 U/L, and calculated MELD score of 13. The physician suspectedthat the patient's hepatic function may be deteriorating and wasconcerned about possible accumulation of PAA. She calculated the ratioof PAA to PAGN as 2.4, and confirmed that the patient had notexperienced any unusual symptoms such as dizziness, headache, or nausea.Considering patient's ammonia control, lack of specific side effects,and clinical remission, the physician decided not to change the dose andto see the patient in two weeks to repeat the laboratory tests. Thephysician also warned the patient to call her immediately if heexperienced any of these symptoms. In two weeks, the patient'slaboratory assessments were essentially unchanged from the previousvisit, with a PAA to PAGN ratio of 2.3, and the patient did not reportany unusual symptoms. Based on the PAA:PAGN ratio of less than 2.5, thephysician decided to continue dosing with 6 mL BID of HPN-100 until thenext routine visit.

Example 9 Analysis of PAA:PAGN Ratio as a Guide to Monitoring Treatmentin a Patient With Parkinson's Disease

HPN-100 treatment was initiated at a dose of 4 mL twice a day in apatient with Parkinson's Disease to produce target circulating levels ofPAA expected to produce clinical benefit. After one week of treatment,the patient's circulating PAA level of 50 μg/mL was below the targetrange, and the PAA:PAGN ratio was determined to be 0.9. The physicianconcluded that the HPN-100 dose could be safely adjusted upward, and thedose was increased by 50% to 6 mL BID. The PAA level and PAA/PAGN ratioone week later were found to be 75 μg/mL and 1.4, respectively. Since 75μg/mL was still below the therapeutic PAA target level and the PAA:PAGNratio of 1.4 indicated that conversion of PAA to PAGN had not beensaturated, the patient's dosage was increased again by 50% to 9 mL BID.One week later, the patient's PAA and PAA:PAGN ratio were found to be159 μg/mL and 2.6, respectively. Since the target PAA level was nowapproximately therapeutic but the PAA:PAGN ratio indicated that PAA toPAGN conversion was approaching saturation, HPN-100 dosage was decreasedto 8 mL BID, at which time the patient's circulating PAA level wasdetermined to be close to the target range and his PAA:PAGN ratio wasdetermined to be 2. The patient's dose was not further adjusted and hecontinued to be monitored.

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

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1-13. (canceled)
 14. A method of treating a urea cycle disorder in asubject experiencing symptoms of nausea, headache, somnolence, orconfusion, having a plasma ammonia level within normal limits, andhaving a plasma PAA to PAGN ratio exceeding 2.5, the method comprising:administering to said subject, a dosage of glyceryltri-[4-phenylbutyrate] effective to achieve a plasma PAA to PAGN ratiobelow 2.5.
 15. The method of claim 14, wherein the subject has moderatehepatic impairment.
 16. The method of claim 14, wherein the subject hasa low body surface area.
 17. The method of claim 14, wherein the dosageof glyceryl tri-[4-phenylbutyrate] is administered orally.
 18. Themethod of claim 14, further comprising measuring the plasma ammonialevel of the subject.